The objective of this competing continuation R01 application is to extend understanding of the molecular mechanisms involved in the mineralization of extracellular matrices in bone and dentin through studies of the sequences, biosynthesis, post-translational processing and secretion of the macromolecules which are involved, and their structures and interactions within the extracellular matrix. The working hypothesis is that certain acidic, phosphorylated, matrix macromolecules are localized by an interaction with the collagen I matrix, and then direct the initiation of crystal deposition and regulate crystal growth in terms of orientation of the crystal axes, crystal shape and crystal size. Although applicable to bone, this study is focused on the dentin system and the principal dentin specific macromolecules, namely phosphophoryn (PP), dentin matrix protein 1 (DMP1), dentin matrix protein 2 (DMP2), and dentin sialoprotein (DSP). Recent successes in cloning DMP1, PP and DSP from their rat incisor odontoblast cDNA library have made it possible to prepare recombinant DMP1 and the C-terminal domain of DMP2. At the same time, the Principal Investigator and his colleagues have discovered that there is a family of PPs, perhaps related as differentially spliced gene products. In the next project period studies are proposed to complete the sequencing of the rat incisor DMP2 and other members of the PP family in rat and other species, including human. Further, the structure of PP will be examined by both molecular modeling and direct examination of the rDMP2 (+/- phosphorylation) by FTIR and ORD-CD, and by x-ray diffraction of crystals prepared from specific recombinant domains. The phosphorylation of rDMP2 and native riPP will be studied as a hierarchical process involving several kinases, particularly CK2 isoforms and cellular compartments. The specificity of interaction between collagen I molecules and riPP and rDMP2 domains will be studied as a function of pH, ionic strength and the presence of Ca2+. The Dmp2 gene has been co-localized with the mineralization disorder Dentinogenesis Imperfecta Type II (DI-II) on human chromosome 4q21, and its possible relationship with DI-II will be explored.
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